Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction–liquid chromatography–tandem mass spectrometry

A simple, sensitive and specific automated SPE–LC–MS–MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C18 solid-phase extraction cartridge using a Zymark RapidTrace™ automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile–water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313→118, m/z 329→196 and m/z 343→196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1–200 ng/ml for I and II and 2–200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.